This proposal is to further develop and exploit a novel in vitro mammalian brainstem-spinal cord preparation to study neural mechanisms controlling breathing. The preparation consists of the brainstem and spinal cord isolated from neonatal rat and maintained under controlled in vitro conditions. This preparation retains major components of the central nervous system (CNS) respiratory pattern generating circuitry and spontaneously generates a respiratory motor output pattern of brainstem origin resembling the pattern generated in the neonate in vivo. Unlike the situation in vivo, the in vitro system allows routine application of most current neurobiological techniques to study mechanisms of neural pattern generation, including direct utilization of pharmacological and membrane channel probes in the CNS, and intracellular recording from different brainstem- spinal cord regions to determine neuron electrophysiological properties. Preliminary studies have shown the in vitro brainstem-spinal cord to be a versatile and potentially powerful experimental system for investigation of motor control systems in the mammalian CNS. The in vitro system will be used to investigate the following aspects of respiratory pattern generation: (1) Functional organization of the pattern generation system. Information will be obtained on how brainstem respiratory networks are organized for the generation of the basic components of the respiratory pattern including respiratory cycle timing (rhythm) and the detailed spatiotemporal patterns of motoneuronal activity. (2) Synaptic mechanisms in pattern generation, including the roles of excitatory and inhibitory synaptic interactions and neurotransmitter systems in rhythm generation and generation of brainstem neuronal discharge patterns. (3) Membrane biophysical properties of respiratory neurons. Intracellular recording will be used to examine electrophysiological behavior of identified brainstem respiratory neurons and to infer the nature of their synaptic inputs and the ionic basis of neuronal excitability. The long range goal of this work is to explain the neurogenesis of respiratory pattern in mammals in terms of the biophysical, synaptic and network properties of CNS respiratory neurons. Information to be obtained from this proposal is fundamental for defining CNS mechanisms responsible for respiratory homeostasis and for understanding pathologies where ventilatory failure results from dysfunction of neural mechanisms controlling breathing.